The broad, long-range goal of this study is to determine the molecular mechanism of hearing and balance. Actin-filled projections, or stereocilia, on the sensory hair cells of the inner ear convert force produced by sounds and mechanical vibrations into nerve impulses. The prevailing model for mechanotransduction, thought to be well-conserved for both cochlear and vestibular hair cells, is one in which neighboring stereocilia connected by extracellular filaments called tip links, deflect in response to stimuli, thereby causing the opening or closing of transduction channels. The channels are associated with an adaptation-motor complex, which when tension is high, is pulled down the actin cytoskeleton of the stereocilia, reducing tip link tension, and opening the channels; and when tension is low, translocates up the actin cytoskeleton, restoring the resting tension, and closing the Ca2* channels. Localization of the molecular motor, myosin 1c (Myolc) at strategic places in the stereocilia; and studies using transgenic mice expressing a mutant Myolc that can be selectively inhibited have shown Myolc1 s involvement in this process known as adaptation (Holt et al, 2002; Stauffer et al., 2005). Adaptation allows hair cells under prolonged stimuli to remain sensitive to new stimuli. The goal of this proposal is to determine the role of Myolc in specific aspects of adaptation by measuring adaptation in mice expressing Myolc mutants with defined molecular properties. The specific aims are: to characterize the biochemical and mechanical properties of Myolc mutants by in vitro expression followed by characterization by ATPase assays, motility assays, kinetic analyses and single-molecule mechanical studies. Mutants will include those that affect the ability of Myo1 c to adapt to mechanical load, a property predicted for Myolc from previous studies in this laboratory (Batters et al., 2004a; 2004b). Next, the effects of these mutations on adaptation will be tested in vestibular hair cells from knockin mice expressing mutant Myolc. The combined in vitro and animal studies are expected to provide critical, new insight into the molecular mechanism of Myolc and its role in adaptation. This knowledge could ultimately lead to the design of rational diagnostics and therapies to treat diseases of balance and/or hearing.